Process for the preparation of 6,7-diacyl-7-deacetylforskolin derivatives

ABSTRACT

This invention relates to a process for the preparation of a 6,7-diacyl-7-deacetylforskolin derivative represented by the general formula: ##STR1## wherein R 1  and R 2  each stands for an acyl group and R 3  stands for an aliphatic group having 2 to 3 carbon atoms, which comprises eliminating the acyl group at position 1 in a 1,6,7-triacyl-7-deacetylforskolin derivative represented by the general formula: ##STR2## wherein R 1 , R 2  and R 3  are as defined above, by solvolysis. The compound of the formula (I) is expected as medicine.

This application is a division of application Ser. No. 08/179,826, filedJan. 19, 1994, now U.S. Pat. No. 5,484,954 which is a division of Ser.No. 08/071,616, filed Jun. 4, 1993, which is a continuation of Ser. No.07/624,528, filed Dec. 10, 1990, now abandoned, which is acontinuation-in-part of Ser. No. 07/212,013, filed Jun. 27, 1988, nowabandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel process for the preparation ofa 6,7-diacyl-7-deacetylforskolin derivative which is expected as amedicine.

2. Description of the Prior Art

Known processes for the preparation of a 6,7-diacyl-7-deacetylforskolinderivative include a process which comprises directly acylating thehydroxyl group at position 7 of the compound of the general formula (II)(EP222413A).

However, the direct acylation process according to the prior art isdisadvantageous in that the selectivity for the acetylation of thehydroxyl group at position 7 against position 1 is low.

SUMMARY OF THE INVENTION

The inventors of the present invention have eagerly studied and havefound that high-quality 6,7-diacyl-7-deacetylforskolin derivativerepresented by the general formula: ##STR3## wherein R¹ and R² eachstands for an acyl group and R³ stands for an aliphatic group having 2to 3 carbon atoms, or a cyclopropyl group can be unexpectedly preparedby acylating the hydroxy groups at positions 1 and 7 of a6-acyl-7-deacetylforskolin derivative represented by the generalformula: ##STR4## wherein R² and R³ are as defined above, with anacylating agent to obtain a 1,6,7-triacyl-7-deacetylforskolin derivativerepresented by the general formula: ##STR5## wherein R¹, R² and R³ areas defined above, and subjecting the 1,6,7-triacyl-7-deacetylforskolinderivative to solvolysis to thereby selectively remove the acyl group atposition i of the derivative.

The present invention has been accomplished on the basis of thisfinding.

DETAILED DESCRIPTION OF THE INVENTION

Examples of the acyl group of the R¹ in the general formulas (I) and(III) include formyl, acetyl, propionyl, butyryl, pentanoyl, hexanoyl,glycyl, dimethylaminoacetyl, piperidinoacetyl,t-butoxycarbonylaminoacetyl, glycoloyl, 2-morpholinopropionyl,2-aminopropionyl, 3-dimethylaminopropionyl,3-benzyloxycarbonylaminopropionyl, 2,3-dihydroxypropionyl,2-pyrrolidinobutyryl, 3-thiomorpholinobutyryl, 4-dimethylaminobutyryl,5-methylaminopentanoyl, 6-dimethylaminohexanoyl, tyrosyl, hemisuccinyl,thienoyl, prolyl, histidyl, lysyl, ornithyl, 2-dimethylaminopropionyl,3-dimethylamino-2-methylpropionyl and 4-dimethylamino-2-methylbutyrylgroups.

Examples of the acyl group of the R² in the general formulas (I), (II)and (III) include formyl, acetyl, propionyl, butyryl, pentanoyl,hexanoyl, glycyl, dimethylaminoacetyl, piperidinoacetyl,t-butoxycarbonylaminoacetyl, glycoloyl, 2-morpholinopropionyl,2-aminopropionyl, 3-dimethylaminopropionyl,3-benzyloxycarbonylaminopropionyl, 2,3-dihydroxypropionyl,2-pyrrolidinobutyryl, 3-thiomorpholinobutyryl, 4-dimethylaminobutyryl,5-methylaminopentanoyl, 6-dimethylaminohexanoyl, hemisuccinyl, thienoyl,prolyl, histidyl, lysyl, tyrosyl, ornithyl, 2-dimethylaminopropionyl,3-dimethylamino-2-methylpropionyl, 4-dimethylamino-2-methylbutyryl and2,2-dimethyl-1,3-dioxolane-4-carbonyl groups.

Examples of R³ include aliphatic groups having 2 to 3 carbon atoms, suchas vinyl and ethyl or cyclopropyl groups.

Examples of the compound of the general formula (II) include7-deacetyl-6-dimethylaminoacetylforskolin, 7-deacetyl-6-glycylforskolin,7-deacetyl-6-piperidinoacetylforskolin,7-deacetyl-6-(2-dimethylaminopropionyl)forskolin,7-deacetyl-6-(3-dimethylaminopropionyl)forskolin,7-deacetyl-6-(2-morpholinopropionyl)forskolin,7-deacetyl-6-alanylforskolin, 7-deacetyl-6-(2-aminobutyryl)forskolin,7-deacetyl-6-(4-dimethylaminobutyryl)forskolin,7-deacetyl-6-(2,3-dihydroxypropionyl)forskolin,7-deacetyl-6-hemisuccinylforskolin, 7-deacetyl-6-histidylforskolin,7-deacetyl-6-prolylforskolin, 7-deacetyl-6-lysylforskolin,7-deacetyl-6-glycoloylforskolin,7-deacetyl-14,15-dihydro-6-dimethylaminoacetylforskolin,7-deacetyl-14,15-dihydro-6-(3-dimethylaminopropionyl)forskolin,7-deacetyl-14,15-dihydro-6-(4-dimethylaminobutyryl)forskolin,13-cyclopropyl-7-deacetyl-6-(3-dimethylaminopropionyl)-14,15-dinorforskolin,13-cyclopropyl-7-deacetyl-6-(4-dimethylaminobutyryl)-14,15-dinorforskolin,7-deacetyl-14,15-dihydro-6-piperidinoacetylforskolin,7-deacetyl-14,15-dihydro-6-(2-morpholinopropionyl)forskolin,7-deacetyl-6-(t-butoxycarbonylaminoacetyl)forskolin,7-deacetyl-6-(2-benzyloxycarbonylaminopropionyl)forskolin,7-deacetyl-6-(2-t-butoxycarbonylaminobutyryl)forskolin,7-deacetylforskolin-6-(2,2-dimethyl-1,3-dioxolane-4-carboxylate),7-deacetyl-6-(3-methoxycarbonylpropionyl)forskolin,7-deacetyl-6-(3-dimethylamino-2-methylpropionyl)forskolin,7-deacetyl-14,15-dihydro-6-(3-dimethylamino-2-methylpropionyl)forskolin,7-deacetyl-6-(4-dimethylamino-2-methylbutyryl)forskolin,7-deacetyl-14,15-dihydro-6-(4-dimethylamino-2-methylbutyryl)forskolin,13-cyclopropyl-7-deacetyl-6-(3-dimethylamino-2-methylpropionyl)-14,15-dinorforskolinand13-cyclopropyl-7-deacetyl-6-(4-dimethylamino-2-methylbutyryl)-14,15-dinorforskolin.

Examples of the compound represented by the general formula (III)include 1-formyl-6-dimethylaminoacetyl-7-deacetyl-7-formylforskolin,1-acetyl-6-dimethylaminoacetylforskolin,1-propionyl-6-dimethylaminoacetyl-7-deacetyl-7-propionylforskolin,1-acetyl-6-piperidinoacetylforskolin,1-butyryl-6-piperidinoacetyl-7-deacetyl-7-butyrylforskolin,1-acetyl-6-(2-dimethylaminopropionyl)forskolin,1-pentanoyl-6-(2-dimethylaminopropionyl)-7-deacetyl-7-pentanoylforskolin,1-acetyl-6-(3-dimethylaminopropionyl)forskolin,1-hexanoyl-6-(3-dimethylaminopropionyl)-7-deacetyl-7-hexanoylforskolin,1-dimethylaminoacetyl-6-(3-dimethylaminopropionyl)-7-deacetyl-7-dimethylaminoacetylforskolin,1-piperidinoacetyl-6-(3-dimethylaminopropionyl)-7-deacetyl-7-piperidinoacetylforskolin,1-(t-butoxycarbonylaminoacetyl)-6-(3-dimethylaminopropionyl)-7-deacetyl-7-(t-butoxycarbonylaminoacetyl)forskolin,1-acetyl-6-(2-morpholinopropionyl)forskolin,1-(2-morpholinopropionyl)-6-(2-morpholinopropionyl)-7-deacetyl-7-(2-morpholinopropionyl)forskolin,1-acetyl-6-(4-dimethylaminobutyryl)forskolin,1-(3-dimethylaminopropionyl)-6-(4-dimethylaminobutyryl)-7-deacetyl-7-(3-dimethylaminopropionyl)forskolin,1-(3-benzyloxycarbonylaminopropionyl)-6-(4-dimethylaminobutyryl)-7-deacetyl-7-(3-benzyloxycarbonylaminopropionyl)forskolin,1-(2-pyrrolidinobutyryl)-6-(4-dimethylaminobutyryl)-7-deacetyl-7-(2-pyrrolidinobutyryl)forskolin,1-acetyl-6-hemisuccinylforskolin,1-(3-thiomorpholinobutyryl)-6-hemisuccinyl-7-deacetyl-7-(3-thiomorpholinobutyryl)forskolin,1-acetyl-14,15-dihydro-6-dimethylaminoacetylforskolin,1-(4-dimethylaminobutyryl)-14,15-dihydro-6-dimethylaminoacetyl-7-deacetyl-7-(4-dimethylaminobutyryl)forskolin,1-acetyl-14,15-dihydro-6-(3-dimethylaminopropionyl)forskolin,1-(6-dimethylaminohexanoyl)-14,15-dihydro-6-(3-dimethylaminopropionyl)-7-deacetyl-7-(6-dimethylaminohexanoyl)forskolin,1-acetyl-14,15-dihydro-6-(4-dimethylaminobutyryl)forskolin,1-propionyl-14,15-dihydro-6-(4-dimethylaminobutyryl)-7-deacetyl-7-propionylforskolin,1-acetyl-13-cyclopropyl-6-(3-dimethylaminopropionyl)-14,15-dinorforskolin,1-acetyl-13-cyclopropyl-6-(4-dimethylaminobutyryl)-14,15-dinorforskolin,1-acetyl-14,15-dihydro-6-piperidinoacetylforskolin,1-acetyl-14,15-dihydro-6-(2-morpholinopropionyl)forskolin,1-acetyl-6-(3-dimethylamino-2-methylpropionyl)forskolin,1-acetyl-14,15-dihydro-6-(3-dimethylamino-2-methylpropionyl)forskolin,1-acetyl-13-cyclopropyl-6-(3-dimethylamino-2-methylpropionyl)-14,15-dinorforskolin,1-acetyl-6-(4-dimethylamino-2-methylbutyryl)forskolin,1-acetyl-14,15-dihydro-6-(4-dimethylamino-2-methylbutyryl)forskolin and1-acetyl-13-cyclopropyl-6-(4-dimethylamino-2-methylbutyryl)-14,15-dinorforskolin.

Examples of the acylating agent to be used in the present inventioninclude formic acid, acetic acid, propionic acid, butyric acid, valericacid, caproic acid, glycine, N,N-dimethylglycine, piperidinoacetic acid,N-t-butoxycarbonylglycine, glycolic acid, 2-morpholinopropionic acid,alanine, 3-dimethylaminopropionic acid,3-benzyloxycarbonylaminopropionic acid, 2,3-dihydroxypropionic acid,2-pyrrolidinobutyric acid, 3-thiomorpholinobutyric acid,4-dimethylaminobutyric acid, 5-methylaminovaleic acid,6-dimethylaminocaproic acid, tyrosine, succinic acid, proline,histidine, lysine, ornithine, thiophenecarboxylic acid,2,2-dimethyl-1,3-dioxolane-4-carboxylic acid,3-dimethylamino-2-methylpropionic acid, 4-dimethylamino-2-methylbutyricacid and reactive derivatives thereof.

Such a reactive derivative includes acid halides, acid anhydrides, mixedacid anhydrides and Leuch's anhydrides.

The acylation of the compound of the general formula (II) is carried outby the use of about 2 to about 50 mol, preferably about 2 to about 4 molof an acylating agent per mol of the compound in a solvent at atemperature of about 0° C. to a boilihg point of the solvent, preferablyabout 0° C. to room temperature for several minutes to about 24 hours,preferably several minutes to about 2 hours. Examples of the solvent tobe used in the acylation include benzene, chloroform, ether,dichloromethane, 1,1,1-trichloroethane, 1,2-dichloroethane, carbontetrachloride and ethyl acetate.

The selective deacylation at position 1 of the compound of the generalformula (III) prepared by the above acylation at position 1 is carriedout by any process selected from among alcoholysis, aminolysis andhydrolysis, preferably by hydrolysis. The hydrolysis is carried out inthe presence of a hydrolyzing agent in a solvent at a temperature ofabout 0° C. to a boiling point of the solvent, preferably about 0° C. toroom temperature for about one minute to about 12 hours, preferablyabout 5 minutes to about 3 hours. It is preferred that the reaction timebe short, when the reaction temperature is high, while the time be long,when the temperature is low. Examples of the solvent to be used in thehydrolysis include water soluble solvent such as alcohol (methanol,ethanol, propanol, isopropanol, butanol, etc.), acetonitrile, dimethyl,sulfoxide, N,N-dimethylformamide, acetone, tetrahydrofuran and mixturesthereof with water up to 50 v/v %, preferably aqueous C₁ -C₄ alcoholcontaining about 0.5 to about 20 v/v % of water. The hydrolyzing agentis a base or an acid.

The base to be used in the hydrolysis include alkali hydroxides such assodium hydroxide and potassium hydroxide; alkali carbonates such aspotassium carbonate; alkali bicarbonates such as sodium bicarbonate andamines such as ammonia and triethylamine, while the acid include mineralacids such as sulfuric and hydrochloric acids. Such a base or acid iscontained in a solvent with a concentration of about 0.01 to about 50%,preferably about 0.05 to about 10 %.

When any of R¹ and R² is a propionyl group having an amino substituentat β-position, for example, a dimethylaminopropionyl group, it ispreferred that the reaction mixture after the acylation or after theselective deacylation be treated with an amine corresponding to theamino substituent in a solvent at a temperature of 0° C. to roomtemperature for one minute to 12 hours to thereby inhibit the formationof a by-product. Examples of the solvent to be used in this treatmentinclude benzene, carbon tetrachloride, dichloromethane, chloroform,1,2-dichloroethane, 1,1,1-trichloroethane, ether, diisopropyl ether,ethyl acetate, acetone, propanol, ethanol and methanol.

Further, the above amine may be used in an amount of about 0.1 to about3 mol per mole of the compound of the general formula (II) used.

If circumstances requires, excesses of selective deacetylation arises toobtain compound (II). But the excesses of the reaction can be avoided byneutrelization after the completion of selective deacetylation.

After the completion of the reaction, a 6,7-diacyl-7-deacetylforskolinderivative thus prepared can be isolated by concentrating the reactionmixture, optionally diluting the obtained residue with water or anaqueous solution of common salt, extracting the obtained mixture with anorganic solvent, washing the organic layer, drying and concentrating thewashed layer and recrystallizing the obtained residue. Thus, accordingto the present invention, the desired compound can be easily purifiedwithout employing chromatography or the like.

The yields of the objective compounds prepared by the process of thepresent invention (Example 1-1 or 1-2) and by the process described inE.P. 222413A are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                               Yield                                                                         6-(4-dimethylamino-                                                                         6-(3-dimethylamino-                                             butyryl)-forskolin                                                                          propionyl)-forskolin                                            [yield based on                                                                             [yield based on                                                 7-deacetyl-6-(4-dimethyl-                                                                   7-deacetyl-6-(3-dimethyl-                                       aminobutyryl) aminopropionyl)                                                 forskolin]    forskolin]                                               ______________________________________                                        The process                                                                              83%    (Ex. 1-1)    54%  (Ex. 1-2)                                 of the                                                                        present                                                                       invention                                                                     The process                                                                              31%                 32%                                            of the                                                                        prior art                                                                     ______________________________________                                    

It is apparent from the results shown in the above table that a6,7-diacyl-7-deacetylforskolin can be prepared by the process of thepresent invention in a high yield.

The process described in E.P. 222413A mentioned above is as follows:

A solution of 6-acyl-7-deacetylforskolin in dichloromethane is reactedwith acetyl chloride in the presence of pyridine. After the completionof the reaction, the reaction product is purified by silica gelchromatography.

EXAMPLE 1-1

6-(4-Dimethylaminobutyryl)forskolin

30 ml of acetic anhydride was added to a mixture comprising 31.7 g of7-deacetyl-6-(4-dimethylaminobutyryl)forskolin, 0.5 g of4-dimethylaminopyridine and 120 ml of pyridine under cooling with ice.The mixture was stirred at room temperature for two days. After thecompletion of the reaction, the reaction mixture was concentrated.

The concentrate was dissolved in 600 ml of methanol, followed by theaddition of 250 ml of 1N aqueous sodium hydroxide. The mixture wasstirred at a room temperature for 35 minutes and filtered to give 31.2 gof a crude crystal. This crude crystal was recrystallized from acetoneto give 28.7 g of 6-(4-dimethylaminobutyryl)forskolin [yield based on7-deacetyl-6-(4-dimethylaminobutyryl)forskolin: 83%].

IR(KBr)ν: 3450, 1730, 1710 cm⁻¹.

The reaction product before the hydrolysis in the above synthesis waspurified by silica gel column chromatography to obtain1-acetyl-6-(4-dimethylaminobutyryl)forskolin.

¹ H-NMR (CDCl₃)δ: 5.84 (1H, t, J=3.5 Hz), 5.57 (1H, brs), 5.56 (1H, d,J=4.8 Hz), 2.41 (6H, s), 2.03 (6H, s), 1.64 (3H, s), 1.52 (3H, s), 1.34(3H, s), 1.04 (3H, s), 0.97 (3H, s).

EXAMPLE 1-2

6-(3-Dimethylaminopropionyl)forskolin

A mixture comprising 12 g of7-deacetyl-6-(3-dimethylaminopropionyl)forskolin, 35 mg of4-dimethylaminopyridine, 48 ml of anhydrous pyridine and 5.9 ml ofacetic anhydride was stirred at room temperature for 2 hours, followedby the addition of 35 mg of 4-dimethylaminopyridine. The mixture wasfurther stirred for 3 hours. After the completion of the reaction, 5 mlof methanol was added to the reaction mixture and the mixture wasconcentrated.

The concentrate was diluted with 120 ml of methanol and 25 ml of water,followed by the addition of 40 ml of 1N aqueous sodium hydroxide. Themixture was stirred at room temperature for 30 minutes. 50 ml of 1Nhydrochloric acid was added to the mixture to stop the reaction and themixture was concentrated. The concentrate was diluted with water,basified with concentrated aqueous ammonia and extracted withdiisopropyl ether. The organic layer was dried over anhydrous magnesiumsulfate and filtered to remove the drying agent. The filtrate wasconcentrated to give 13.4 g of a residue. This residue was dissolved indichloromethane, followed by the addition of dimethylamine. The mixturewas stirred at a room temperature for one hour. After the completion ofthe reaction, the reaction mixture was concentrated to give a residue.This residue was recrystallized from ethanol and then fromcyclohexane/dichloromethane to give 7.04 g of6-(3-dimethylaminopropionyl)forskolin [yield based on7-deacetyl-6-(3-dimethylaminopropionyl)forskolin: 54%].

IR(KBr)ν: 3200, 1735, 1710 cm⁻¹.

The reaction product obtained before the hydrolysis in the abovesynthesis of 6-(3-dimethylaminopropionyl)forskolin was purified bysilica gel column chromatography to give1-acetyl-6-(3-dimethylaminopropionyl)forskolin.

IR(Nujol)ν: 3500, 1735, 1710 cm⁻¹.

¹ H-NMR (CDCl₃)δ: 5.85 (1H, q, J=4.8 Hz, J=2.9 Hz), 5.57 (1H, brs), 5.55(1H, d, J=4.8 Hz), 2.32 (6H, s), 2.023 (3H, s), 2.020 (3H, s), 1.65 (3H,s), 1.53 (3H, s), 1.34 (3H, s), 1.04 (3H, s), 0.98 (3H, s).

MS (hydrochloride) m/z (relative intensity): 552 (M⁺ of free base, 18),160 (22), 118 (70), 116 (33), 59 (39), 58 (100).

EXAMPLE 1-3

1-Acetyl-6-(2-morpholinopropionyl)forskolin

A solution of 945 mg of acetyl chloride in dichloromethane was added insix portions to a mixture comprising 700 mg of7-deacetyl-6-(2-morpholinopropionyl)froskolin, 1 g of pyridine and 30 mlof dichloromethane at room temperature over a period of 9 hours. Afterthe completion of the reaction, water was added to the reaction mixtureand the mixture was basified with 20% aqueous sodium hydroxide andextracted with dichloromethane. The organic layer was dried overanhydrous magnesium sulfate and filtered to remove the drying agent. Thefiltrate was concentrated to give a residue. This residue wasrecrystallized from hexane/toluene to obtain 606 mg of1-acetyl-6-(2-morpholinopropionyl)forskolin as a colorless soliddiastereomeric mixture (yield: 75%).

IR(Nujol)ν: 3490, 1750, 1715, 1230 cm⁻¹.

MS m/z (relative intensity): 593 (M⁺, 0.03), 549 (0.7), 518 (0.5), 410(0.7), 228 (0.9), 191 (1.2), 160 (8), 115 (23), 114 (100), 70 (9).

6-(2-Morpholinopropionyl)forskolin

0.75 ml of 1N aqueous sodium hydroxide was added to a mixture comprising372 mg of 1-acetyl-6-(2-morpholinopropionyl)forskolin and 20 ml ofmethanol. The mixture was stirred at room temperature for 30 minutes.After the completion of the reaction, the reaction mixture wasconcentrated. The concentrate was diluted with a saturated aqueoussolution of common salt and extracted with ethyl acetate. The organiclayer was dried over anhydrous magnesium sulfate and filtered to removethe drying agent. The filtrate was concentrated to give 388 mg of asolid residue. This residue was recrystallized from hexane/ethyl acetateto give 214 mg of 6-(2-morpholinopropionyl)forskolin (yield: 62%).

IR(Nujol)ν: 3170, 1735, 1710 cm⁻¹.

MS m/z (relative intensity): 551 (M⁺, 0.2), 518 (2), 158 (20), 115(100), 114 (100), 70 (47).

EXAMPLE 1-4

6-Piperidinoacetylforskolin

520 mg of acetic anhydride was added to a mixture comprising 188 mg of7-deacetyl-6-piperidinoacetylforskolin, 10 mg of 4-dimethylaminopyridineand 4 ml of pyridine under cooling with ice. The mixture was stirred ata room temperature for 3.5 hours. After the completion of the reaction,methanol was added to the reaction mixture under cooling with ice. Themixture was concentrated to give an oily residue. A saturated aqueoussolution of common salt was added to the residue. The obtained mixturewas basified with concentrated aqueous ammonia and extracted with ethylacetate. The organic layer was dried over anhydrous magnesium sulfateand filtered to remove the drying agent. The filtrate was concentratedto give 224 mg of a solid product. 120 mg of this product was dissolvedin 5 ml of methanol, followed by the addition of 0.20 ml of 1N aqueoussodium hydroxide. The mixture was stirred at a room temperature for 25minutes. After the completion of the reaction, the reaction mixture wasconcentrated. The concentrate was diluted with a saturated aqueoussolution of common salt and extracted with ethyl acetate. The organiclayer was dried over anhydrous magnesium sulfate and filtered to removethe drying agent. The filtrate was concentrated to obtain 113 mg of aresidue. This residue was recrylstallized from hexane/ethyl acetate toobtain 59 mg of 6-piperidinoacetylforskolin [yield based on7-deacetyl-6-piperidinoacetylforskolin: 54%].

IR(Nujol)ν: 3180, 1745, 1710 cm⁻¹

¹ H-NMR (CDCl₃)δ: 5.85 (1H, q, J=4.2 Hz, J=2.7 Hz), 5.55 (1H, d, J=4.4Hz), 4.61 (1H, br, s), 3.18 and 3.14 (each 1H, d, J=16 Hz), 2.5 (4H, m),2.03 (3H, s), 1.66 (3H, s), 1.44 (3H, s), 1.35 (3H, s), 1.03 (3H, s),0.96 (3H, s)

MS m/z: 535 (M⁺)

The reaction product before the selective hydrolysis with sodiumhydroxide in the above synthesis was purified by silica gel columnchromatography to give 1-acetyl-6-piperidinoacetylforskolin.

MS m/z: 577 (M⁺)

EXAMPLE 1-5

6-(3-Dimethylaminopropionyl)-14,15-dihydroforskolin hydrochloride

A mixture comprising 200 mg of7-deacetyl-6-(3-dimethylaminopropionyl)-14,15-dihydroforskolin, 7 mg of4-dimethylaminopyridine, 2 ml of anhydrous pyridine and 0.4 ml of aceticanhydride was stirred at a room temperature for 3 hours. After thecompletion of the reaction, 1 ml of methanol was added to the reactionmixture under cooling with ice and the mixture was concentrated.

The concentrate was diluted with a saturated aqueous solution of commonsalt, basified with concentrated aqueous ammonia, and extracted withethyl acetate. The organic layer was dried over anhydrous magnesiumsulfate and filtered to remove the drying agent. The filtrate wasconcentrated to obtain1-acetyl-6-(3-dimethylaminopropionyl)-14,15-dihydroforskolin (231 mg).

The concentrate was diluted with 2 ml of methanol, followed by theaddition of 0.4 ml of 0.25N aqueous sodium hydroxide under cooling withice. The mixture was stirred under cooling with ice for 1.5 hours. 0.6ml of 1N hydrochloric acid was added to the mixture to stop the reactionand the mixture was concentrated. The concentrate was diluted withwater, basified with concentrated aqueous ammonia and extracted withethyl acetate. The organic layer was dried over anhydrous magnesiumsulfate and filtered to remove the drying agent. The filtrate wasconcentrated to give 202 mg of a residue. This residue was dissolved in2 ml of dichloromethane, followed by the addition of 0.15 ml of 4Nhydrochloric acid in dioxane. The mixture was concentrated to give 210mg of a solid residue. This residue was recrystallized frommethanol-ethyl acetate (2:3) to give 112 mg of6-(3-dimethylaminopropionyl)-14,15-dihydroforskolin hydrochloride [yieldbased on 7-deacetyl-6-(3-dimethylaminopropionyl)-14,15-dihydroforskolin:48%].

Reference Example 1

7-Deacetyl-6-(3-dimethylaminopropionyl)-14,15-dihydroforskolin

A mixture of 703 mg of 7-deacetyl-6-(3-dimethylaminopropionyl)forskolin,23 mg of 5% palladium on carbon catalyst, and 20 ml of methanol wasstirred under a hydrogen atmosphere at room temperature for 1 hour and40 minutes to complete the reaction. The reaction mixture was filteredto remove the catalyst. The filtrate was concentrated to obtain 689 mgof a solid residue. This residue was recrystallized fromchloroform-isopropyl ether (1:5) to obtain 541 mg of7-deacetyl-6-(3-dimethylaminopropionyl)-14,15-dihydroforskolin (yield:77%).

m.p. 163°-165° C.

IR(Nujol)ν: 3200, 1735, 1705 cm⁻¹

¹ H-NMR (CDCl₃)δ: 5.94 (1H, t, J=4 Hz), 4.50 (1H, brs), 4.18 (1H, d, J=5Hz), 2.26 (6H, s), 1.53 (3H, s), 1.42 (3H, s), 1.31 (3H, s), 1.10 (3H,s), 0.97 (3H, t, J=7 Hz), 0.95 (3H, s).

MS m/z (relative intensity): 469 (M⁺, 5), 159 (13), 118 (48), 116 (12),91 (30), 69 (13), 59 (10), 58 (100), 57 (11), 55 (13).

The second invention of the present invention relates to a novel processfor the preparation of a 6-acyl-7-deacetylforskolin derivative which isnoted as a medicine, or starting material of the first invention.

Known processes for the preparation of a 6-acyl-7-deacetylforskolininclude a process comprising treating 7-acyl-7-deacetylforskolin with analkali in a solvent at room temperature to thereby carry out therearrangement of the acyl group at position 7 to position 6 (see E.P.No. 222413A).

In the above process of the prior art, 7-deacetylforskolin is formed bythe hydrolysis of 7-acyl-7-deacetylforskolin as a by-product. Therefore,the process is problematic in yield and is disadvantageous in that aproduct obtained by the process must be purified by chromatography, thusbeing unsuitable for large scale production.

The inventors of the present invention have eagerly investigated andhave found that a 6-acyl-7-deacetylforskolin derivative represented bythe general formula: ##STR6## wherein R¹⁰ stands for a hydroxy groupwhich may be esterified; R² stands for an acyl group and R³ stands foran aliphatic group having 2 to 3 carbon atoms, or a cyclopropyl groupcan be prepared, without using any alkali, only by heating a7-acyl-7-deacetylforskolin derivative represented by the generalformula: ##STR7## wherein R¹⁰, R² and R³ are as defined above to therebycarry out the rearrangement of the acyl group at position 7 to position6.

The R¹⁰ of the above general formulas (1) and (2) includes a hydroxygroup; acyloxy groups such as formyloxy, acetoxy, propionyloxy,butyryloxy, benzoyloxy, 4-methoxybenzoyloxy, dimethylaminoacetoxy,piperidinoacetoxy, diethylaminoacetoxy, morpholinoacetoxy,(4-hydroxypiperidino)acetoxy, dipropylaminoacetoxy,2-ethylaminopropionyloxy, thiomorpholinoacetoxy,2-morpholinopropionyloxy, isopropylaminoacetoxy,2-dimethylaminopropionyloxy, t-butylaminoacetoxy,3-dimethylaminopropionyloxy, (4-methylpiperazino)acetoxy,2-dimethylaminobutyryloxy, 3-dimethylaminobutyryloxy,4-dimethylaminobutyryloxy, glycoloyloxy, 2,3-dihydroxypropionyloxy,thioglycoloyloxy, hemisuccinyloxy, hemiglutaryloxy, glycyloxy,2-aminopropionyloxy, 3-aminopropionyloxy, 2-methylaminobutyryloxy,nicotinoyloxy, furoyloxy, histidyloxy and lysyloxy; silyloxy groups suchas trimethylsilyloxy, t-butyldiphenylsilyloxy andt-butyldimethylsilyloxy groups; substituted alkoxy groups such as2-methoxyethoxymethoxy, methoxy, methylthiomethoxy, methoxymethoxy andbenzyloxy groups and substituted alkoxycarbonyloxy groups such asbenzyloxycarbonyloxy and t-butoxycarbonyloxy groups.

The R² includes formyl, acetyl, propionyl, butyryl, dimethylaminoacetyl,butylaminoacetyl, diethylaminoacetyl, pyrrolidinoacetyl,piperazinoacetyl, morpholinoacetyl, piperidinoacetyl,N-cyclohexyl-N-methylaminoacetyl, (4-methylpiperazino)acetyl,dipropylaminoacetyl, (4-hydroxypiperidino)acetyl, thiomorpholinoacetyl,isopropylaminoacetyl, t-butylaminoacetyl, glycyl,benzyloxycarbonylaminoacetyl, 2-aminopropionyl, 3-aminopropionyl,2-dimethylaminopropionyl, 3-dimethylaminopropionyl,2-pyrrolidinopropionyl, 3-piperazinopropionyl, 2-butylaminopropionyl,3-diethylaminopropionyl, 2-morpholinopropionyl, 3-piperidinopropionyl,3-(t-butoxycarbonylamino)propionyl, 2-aminobutyryl, 3-aminobutyryl,4-dimethylaminobutyryl, 4-aminobutyryl, 2-dimethylaminobutyryl,3-diethylaminobutyryl, 4-isopropylaminobutyryl, 2-butylaminobutyryl,3-pyrrolidinobutyryl, 4-morpholinobutyryl, 2-piperazinobutyryl,3-piperidinobutyryl, 4-thimorpholinobutyryl, 2-aminopentanoyl,3-dimethylaminopentanoyl, 4-diethylaminopentanoyl,5-pyrrolidinopentanoyl, 2-piperidinohexanoyl, 3-morpholinohexanoyl,4-(4-methylpiperazino)hexanoyl, 5-(6-butylamino)hexanoyl,6-methylaminohexanoyl, 3-dimethylamino-2-methylpropionyl,3-pyrrolidino-2-methylpropionyl, 3-dimethylamino-2-ethylpropionyl,4-dimethylamino-2-methylbutyryl, 4-amino-2-propylbutyryl, hemisuccinyl,hemiglutaryl, thioglycoloyl, thienoyl, isonicotinoyl, prolyl, histidyl,lysyl, tyrosyl, methionyl, ornithyl, glycoloyl, lactoyl and2,3-dihydroxypropionyl groups.

Examples of the R³ group include aliphatic groups having 2 to 3 carbonatoms, such as vinyl and ethyl, or a cyclopropyl group.

Examples of the compound represented by the general formula (2) include7-deacetyl-7-dimethylaminoacetylforskolin, 7-deacetyl-7-glycylforskolin,7-deacetyl-7-piperidinoacetylforskolin,7-deacetyl-7-(2-dimethylaminopropionyl)forskolin,7-deacetyl-7-(3-dimethylaminopropionyl)forskolin,7-deacetyl-7-(2-morpholinopropionyl)forskolin,7-deacetyl-7-alanylforskolin, 7-deacetyl-7-(2-aminobutyryl)forskolin,7-deacetyl-7-(4-dimethylaminobutyryl)forskolin,7-deacetyl-7-(2,3-dihydroxypropionyl)forskolin,7-deacetyl-7-hemisuccinylforskolin, 7-deacetyl-7-histidylforskolin,7-deacetyl-7-propionylforskolin, 7-deacetyl-7-lysylforskolin,7-deacetyl-7-glycoloylforskolin,7-deacetyl-14,15-dihydro-7-dimethylaminoacetylforskolin,7-deacetyl-14,15-dihydro-7-(3-dimethylaminopropionyl)forskolin,7-deacetyl-14,15-dihydro-7-(4-dimethylaminobutyryl)forskolin,13-cyclopropyl-7-deacetyl-7-(3-dimethylaminopropionyl)-14,15-dinorforskolin,13-cyclopropyl-7-deacetyl-7-(4-dimethylaminobutyryl)-4,15-dinorforskolin,7-deacetyl-14,15-dihydro-7-pyrrolidinoacetylforskolin,7-deacetyl-14,15-dihydro-7-(2-morpholinopropionyl)forskolin,1-acetylforskolin, 1-t-butyldimethylsilylforskolin, 1-benzoylforskolin,1-benzylforskolin, 1-methylforskolin, 1-trimethylsilylforskolin,1-t-butyldiphenylsilylforskolin, 1-(2-methoxyethoxymethyl)forskolin,1-methylthiomethylforskolin, 1-methoxymethylforskolin,1-benzyloxycarbonylforskolin, 1-(t-butoxycarbonyl)forskolin,1-acetyl-7-deacetyl-7-propionylforskolin,1-t-butyldimethylsilyl-7-butyryl-7-deacetylforskolin,1-benzyl-7-deacetyl-7-pentanoylforskolin,7-deacetyl-7-hexanoyl-1-(2-methoxyethoxymethyl)forskolin,1-benzoyl-14,15-dihydroforskolin,14,15-dihydro-1-trimethylsilylforskolin,1-methoxy-14,15-dihydro-7-deacetyl-7-propionylforskolin,1-t-butyldiphenylsilyl-13-cyclopropyl-14,15-dionor-7-deacetyl-7-butyrylforskolin,1-benzyl-13-cyclopropyl-14,15-dinor-7-deacetyl-7-(3-dimethylaminopropionyl)forskolin,1-(t-butoxycarbonyl)-13-cyclopropyl-14,15-dinor-7-deacetyl-7-(4-dimethylaminobutyryl)forskolin,1-methylthiomethyl-13-cyclopropyl-14,15-dinor-7-deacetyl-7-dimethylaminoacetylforskolin,14,15-dihydroforskolin, 13-cyclopropyl-14,15-dinorforskolin,7-deacetylforskolin-7-(2,2-dimethyl-1,3-dioxolane-4-carbonyl),7-deacetyl-7-(3-dimethylamino-2-methylpropionyl)forskolin,14,15-dihydro-7-deacetyl-7-(3-dimethylamino-2-methylpropionyl)forskolin,7-deacetyl-7-(4-dimethylamino-2-methylbutyryl)forskolin and14,15-dihydro-7-deacetyl-7-(4-dimethylamino-2-methylbutyryl)forskolin.

According to the present invention, in case of using no solvent, thecompound of the general formula (2) must be heated to at least 100° C.,preferably 130° to 300° C., still preferably 150° to 250° C. In case ofusing solvent, reaction temperature is at least about 50° C., preferablyabout 70° C. to boiling point of the solvent.

Although the heating time varies depending upon the temperature, it isgenerally at least 1 minute, preferably 5 minutes to 10 hours, stillpreferably 15 to 60 minutes in case of using no solvent, and 3 to 8hours in case of using solvent.

The method for heating the compound of the general formula (2) is notparticularly limited and the heating of the compound may be carried outeither directly or in a solvent. In the direct heating, therearrangement is preferably carried out in a molten state. On the otherhand, in the heating using a solvent, although the solvent may be eitherone in which the compound of the formula (2) is soluble or one in whichthe compound is insoluble, the solvent is preferably an anhydrous one inorder to inhibit the deacylation of the compound caused by hydrolysis.Preferred examples of the anhydrous solvent includeN,N-dimethylformamide, dimethyl sulfoxide, sulfolane,1-methyl-2-pyrrolidinone, 1,1,2,2-tetrachloroethane, nitrobenzene,chlorobenzene, liquid paraffin, 1,1,2,2-tetrachloroethylene, octylacetate, diglyme, triglyme, acetic acid, pyridine, acetonitrile,triethylamine, and butyronitrile o

After the completion of the reaction, the reaction mixture is cooled toroom temperature and recrystallized to obtain6-acyl-7-deacetylforskolin.

When the R² is a propionyl group having an amino substituent at theβ-position, for example, a 3-dimethylaminopropionyl group, it ispreferred that the reaction mixture after the completion of therearrangement is treated with an amine corresponding to the aminosubstituent at a temperature of 0° C. to room temperature for 1 minuteto 12 hours to thereby inhibit the formation of a by-product. Examplesof the solvent to be used in this treatment include benzene, carbontetrachloride, dichloromethane, chloroform, 1,2-dichloroethane,1,1,1-trichloroethane, ether, diisorpopyl ether, ethyl acetate,acetonitrile, butyronitrile, N,N-dimethylformamide, dimethylsulfoxide,sulfolane, 1-methyl-2-pyrrolidinone, pyridine and acetone. The amount ofthe amine to be used may be 0.1 to 3 mol per mol of the compound of theformula (2) used.

The yield (in terms of pure product) and purity of the objectivecompound obtained by the process according to the present invention(Example 2-1 or 2-4) or by the process of E.P. No. 222413A are shown inTable 2 together with the yield of a by-product (7-deacetylforskolin).

                  TABLE 2                                                         ______________________________________                                                       7-Deacetyl-7-(4-                                                                           7-Deacetyl-7-                                                    dimethyl-    (3-dimethylamino-                                                aminobutyryl)                                                                              propionyl)                                        Starting material                                                                            forskolin    forskolin                                         ______________________________________                                        Ex.   desired compound                                                              yield        84%     (Ex. 2-1)                                                                            74%   (Ex. 2-4)                                   purity*      99.5%          99.7%                                             by-product   0%             0%                                          Comp. desired compound                                                        Ex.   yield        36.9%          52.2%                                             purity*      97%            60%                                               by-product   5%             3%                                          ______________________________________                                         *determined by highperformance liquid chromatography                     

It is apparent from the results shown in the above table 2 that6-acyl-7-deacetylforskolins of high purity can be prepared in a highyield by the process of the present invention without formation of aby-product.

The process of E.P. No. 222413A described above comprises treating7-deacetyl-7-(4-dimethylaminobutyryl)forskolin or7-deacetyl-7-(3-dimethylaminopropionyl)forskolin with sodium hydroxidein an acetonitrile/water (45:55) mixture.

EXAMPLE 2-1

7-Deacetyl-6-(4-dimethylaminobutyryl)forskolin

29.05 g of 7-deacetyl-7-(4-dimethylaminobutyryl)forskolin was heated to160° C., stirred for 10 minutes in a molten state, and then, cooled toroom temperature to give a reaction mixture. This reaction mixture wasrecrystallized from a dichloromethane/methyl ethyl ketone mixture togive 14.71 g of 7-deacetyl-6-(4-dimethylaminobutyryl)forskolin. Therecrystallization mother liquor was concentrated to give a residue. Thisresidue was recrystallized from diisopropyl ether to further give 9.64 gof 7-deacetyl-6-(4-dimethylaminobutyryl)forskolin (total yield: 84%).

IR(KBr)ν: 3150, 1730, 1710 cm⁻¹

EXAMPLE 2-2

7-Deacetyl-6-(4-dimethylaminobutyryl)forskolin

49 mg of 7-deacetyl-7-(4-dimethylaminobutyryl)forskolin was heated to140° C.

Although its melting point is higher than 140° C., it was molten byheating for a short time.

After one hour, the heating was stopped to give 35 mg of7-deacetyl-6-(4-dimethylaminobutyryl)forskolin (yield: 71%).

EXAMPLE 2-3

7-Deacetyl-6-(4-dimethylaminobutyryl)forskolin

A mixture comprising 1.00 g of7-deacetyl-7-(4-dimethylaminobutyryl)forskolin and 15 ml ofN,N-dimethylformamide was stirred under heating at 150° C.

After one hour, the reaction was stopped to give 720 mg of7-deacetyl-6-(4-dimethylaminobutyryrl)forskolin (yield: 72% ).

EXAMPLE 2-4

7-Deacetyl-6-(3-dimethylaminopropionyl)forskolin

40.10 g of 7-deacetyl-7-(3-dimethylaminopropionyl)forskolin was heatedto 150° C. and stirred for 10 minutes in a molten state. The reactionmixture was cooled to room temperature and dissolved in 500 ml ofdichloromethane. Gaseous dimethylamine was introduced into the solutionunder cooling with ice. After the completion of the reaction, thereaction mixture was concentrated to give a solid residue. This residuewas washed with diisopropyl ether to obtain 19.8 g of7-deacetyl-6-(3-dimethylaminopropionyl)forskolin (yield: 49%).

The mother liquor after the washing was concentrated to give 23.41 g ofa residue. This residue was subjected to the same rearrangementtreatment as that described above to further give 9.71 g of7-deacetyl-6-(3-dimethylaminopropionyl)forskolin (total yield: 74%).

EXAMPLE 2-5

7-Deacetyl-6-piperidinoacetylforskolin

145 mg of 7-deacetyl-7-piperidinoacetylforskolin was heated to 180° C.and allowed to stand for 30 minutes in a molten state. The obtainedreaction mixture was cooled and recrystallized from a hexane/ethylacetate mixture to give 83 mg of 7-deacetyl-6-piperidinoacetylforskolin(yield: 57%).

EXAMPLE 2-6

7-Deacetyl-6-(2,3-dihydroxypropionyl)forskolin

190 mg of a mixture of 7-deacetyl-7-(2,3-dihydroxypropionyl)forskolindiastereoisomers was heated to 210° C. and allowed to stand in a moltenstate. After 10 minutes, the reaction was stopped to give a mixture of7-deacetyl-6-(2,3-dihydroxypropionyl)forskolin diastereoisomers.

EXAMPLE 2-7

6-Acetyl-7-deacetylforskolin

120 mg of forskolin was heated to 240° C. and allowed to stand for 5minutes in a molten state. The reaction mixture was cooled andrecrystallized from a hexane/chloroform mixture to give 56 mg of6-acetyl-7-deacetylforskolin (yield: 47%).

EXAMPLE 2-8

7-Deacetyl-1,6-diacetylforskolin

145 mg of 1-acetylforskolin was heated to 240° C. and allowed to standin a molten state.

After 5 minutes, the reaction was stopped to give7-deacetyl-1,6-diacetylforskolin.

EXAMPLE 3-1

7-Deacetyl-6-(3-dimethylaminopropionyl)forskolin

297 mg of 7-deacetyl-7-(3-dimethylaminopropionyl)forskolin, 20 ml ofacetonitrile are mixed and refluxed by heating (81.6° C.). After 4hours, the reaction solution is concentrated under reduced pressure.Obtained residue is diluted with 20 ml of dichloromethane. 2 ml of 50%aqueous dimethylamine solution is added to the solution and stirred for3 hours at room temperature to obtain7-deacetyl-6-(3-dimethylaminopropionyl)forskolin.

EXAMPLE 3-2

1.12 g of 7-deacetyl-7-(3-dimethylaminopropionyl)forskolin and 30 ml ofacetic acid are mixed and refluxed by heating (118° C.) for 1 hour withstirring to obtain 7-deacetyl-6-(3-dimethylaminopropionyl)forskolin.

EXAMPLE 3-3

1.02 g of 7-deacetyl-7-(3-dimethylaminopropionyl)forskolin and 20 ml ofpyridine are mixed and refluxed by heating (115° C.) for 30 hours withstirring to obtain 7-deacetyl-6-(3-dimethylaminopropionyl)forskolin.

EXAMPLE 3-4

394 mg of 7-deacetyl-7-(3-dimethylaminopropionyl)forskolin and 10 ml of1,1,2,2-tetrachrolethane are mixed and stirred for 22 hours at 130° C.to obtain 7-deacetyl-6-(3-dimethylaminopropionyl)forskolin.

EXAMPLE 3-5

7-Deacetyl-6-(3-dimethylaminopropionyl)-14,15-dihydroforskolin

A mixture of 101 mg of7-deacetyl-7-(3-dimethylaminopropionyl)-14,15-dihydroforskolin and 10 mlof acetonitrile was heated under reflux for 6 hours. The reactionmixture was cooled, 0.20 ml of 50% of aqueous dimethylamine was added,and stirred for 2.5 hours at room temperature to obtain7-deacetyl-6-(3-dimethylaminopropionyl)-14,15-dihydroforskolin.

Reference Example 2

7-Deacetyl-7-(3-dimethylaminopropionyl)-14,15-dihydroforskolinhydrochloride

A mixture of 1.00 g of 7-deacetyl-7-(3-dimethylaminopropionyl)forskolin,26 mg of 5% palladium on carbon catalyst, and 20 ml of methanol wasstirred under a hydrogen atmosphere at room temperature for 2 hours tocomplete the reaction. The reaction mixture was filtered to remove thecatalyst. The filtrate was concentrated to obtain an oily residue. Thisresidue was diluted with 5 ml of dichloromethane and 0.6 ml of 4Nhydrochloric acid in dioxane was added. The mixture was concentrated andthe residue was recrystallized from methanol to obtain 339 mg of7-deacetyl-7-(3-dimethylaminopropionyl)-14,15-dihydroforskolinhydrochloride (yield: 31%).

m.p. 260°-261° C. (decomp.)

IR(Nujol)ν: 3230, 2720, 1735, 1710 cm⁻¹,

MS m/z (relative intensity): 469 (M⁺ of free base, 6), 159 (11), 118(41), 116 (12), 86 (16), 84 (25), 71 (11), 69 (11), 58 (100), 57 (16),55 (16).

What is claimed is:
 1. 1,6,7-triacyl-7-deacetylforskolin derivativerepresented by the general formula: ##STR8## wherein R¹ stands for anacyl group, R² stands for 4-dimethylaminobutyryl,3-dimethylaminopropionyl, 2-morpholinopropionyl or piperidinoacetyl andR³ stands for an aliphatic group having 2 to 3 carbon atoms or acyclopropyl group.